(a) Field of the Invention
The invention relates to a method of transforming eukaryotic cells. More particularly, this invention relates to the use of DNA constructs designed to insert a particular DNA fragment more efficiently into host cell's DNA in the goal of transgenesis.
(b) Description of Prior Art
Using recombinant DNA technology, foreign DNA sequences can be inserted into an organism's genome to alter the phenotype of the host's organism. A variety of different procedures have been described and are utilized to produce stably transformed eukaryotic cells. All of these procedures are based on, first introducing the foreign DNA into the eukaryotic cell, and followed by isolation of those cells containing the foreign DNA into the eukaryotic cell's DNA.
Unfortunately, to produce transgenic animal and plant, all current higher eukaryotic cell transformation procedures produce in very low proportions transformed germinal (oocytes, spermatozoa, zygotes, spermatogonia, blastomers, etc.) or stem cells that contain the introduced foreign DNA inserted throughout the genome. Additionally, the random insertion of introduced DNA into the genome of host cells can be lethal if the foreign DNA happens to insert into, and thus mutate, a unique vital native gene in a critical manner.
Introduction of foreign DNA sequence in the mouse and other laboratory animals is now relatively easy to perform and is currently used in transgenesis. At the moment, progress in adapting the technology to higher plants and animals, particularly to commercially exploited animals (e.g. farm animals), has not reached the integration rate of foreign DNA observed in mice. In mice, transgenesis occurs in about six percent of the zygotes injected, whereas it is only 0.6% in pigs, 0.7% in sheep and 0.5% in cattle.
A technique currently exist for selecting cells after homologous recombination (HR) event between an endogenous gene and a DNA construct carrying a copy of the gene (Smitties et al., 1985, Nature, 317:230). This work has been extended for the replacement of a targeted gene or to cause gene deletion as well as introduction of foreign DNA molecules (Thomas et al., 1987, Cell, 51:503). In general, one homologous recombination (HR) event occurs for every 10.sup.2 to 10.sup.5 non-homologous integration events. The HR approach was also tested for mouse transgenesis by microinjecting the DNA construct directly into the fertilized oocytes (Brinster et al., 1989, Proc. Natl. Acad. Sci. USA, 86:7087) The results demonstrated the feasibility of HR to correct a mutant gene which is inactive in fertilized mouse eggs. However, the gene deletion was corrected by HR in one out of five hundred (1/500) transgenic mice that incorporated the injected DNA.
For all the above-mentioned reasons, it would be highly desirable to be provided with a transformation system which would allow integration of a DNA fragment by targeting a repeated site of the host's genome. Preferably, such a system would also provide a means of preventing any insertion into a vital gene or genetic region existing in a single copy.